Deficiency of the BKCa potassium channel displayed significant implications for the physiology of the human bronchial epithelium.

Plasma membrane large-conductance calcium-activated potassium (BKCa) channels are important players in various physiological processes, including those mediated by epithelia. Like other cell types, human bronchial epithelial (HBE) cells also express BKCa in the inner mitochondrial membrane (mitoBKCa). The genetic relationships between these mitochondrial and plasma membrane channels and the precise role of mitoBKCa in epithelium physiology are still unclear. Here, we tested the hypothesis that the mitoBKCa channel is encoded by the same gene as the plasma membrane BKCa channel in HBE cells. We also examined the impact of channel loss on the basic function of HBE cells, which is to create a tight barrier. For this purpose, we used CRISPR/Cas9 technology in 16HBE14o- cells to disrupt the KCNMA1 gene, which encodes the α-subunit responsible for forming the pore of the plasma membrane BKCa channel. Electrophysiological experiments demonstrated that the disruption of the KCNMA1 gene resulted in the loss of BKCa-type channels in the plasma membrane and mitochondria. We have also shown that HBE ΔαBKCa cells exhibited a significant decrease in transepithelial electrical resistance which indicates a loss of tightness of the barrier created by these cells. We have also observed a decrease in mitochondrial respiration, which indicates a significant impairment of these organelles. In conclusion, our findings indicate that a single gene encodes both populations of the channel in HBE cells. Furthermore, this channel is critical for maintaining the proper function of epithelial cells as a cellular barrier.

Changes in Ion Transport across Biological Membranes Exposed to Particulate Matter.

The cells of living organisms are surrounded by the biological membranes that form a barrier between the internal and external environment of the cells. Cell membranes serve as barriers and gatekeepers. They protect cells against the entry of undesirable substances and are the first line of interaction with foreign particles. Therefore, it is very important to understand how substances such as particulate matter (PM) interact with cell membranes. To investigate the effect of PM on the electrical properties of biological membranes, a series of experiments using a black lipid membrane (BLM) technique were performed. L-α-Phosphatidylcholine from soybean (azolectin) was used to create lipid bilayers. PM samples of different diameters (<4 (SRM-PM4.0) and <10 µm (SRM-PM10) were purchased from The National Institute of Standards and Technology (USA) to ensure the repeatability of the measurements. Lipid membranes with incorporated gramicidin A (5 pg/mL) ion channels were used to investigate the effect of PM on ion transport. The ionic current passing through the azolectin membranes was measured in ionic gradients (50/150 mM KCl on cis/trans side). In parallel, the electric membrane capacitance measurements, analysis of the conductance and reversal potential were performed. Our results have shown that PM at concentration range from 10 to 150 µg/mL reduced the basal ionic current at negative potentials while increased it at positive ones, indicating the interaction between lipids forming the membrane and PM. Additionally, PM decreased the gramicidin A channel activity. At the same time, the amplitude of channel openings as well as single channel conductance and reversal potential remained unchanged. Lastly, particulate matter at a concentration of 150 µg/mL did not affect the electric membrane capacity to any significant extent. Understanding the interaction between PM and biological membranes could aid in the search for effective cytoprotective strategies. Perhaps, by the use of an artificial system, we will learn to support the consequences of PM-induced damage.